PROJECT SUMMARY Mycobacterium abscessus complex (MABC) has recently emerged as a significant cause of increasing cases of both community- and hospital-acquired infections, especially among immunosuppressed populations, including populations with cystic fibrosis and transplant patients. This situation is worsened by its exceptionally high natural and acquired antibiotic resistance that complicates treatment, and consequently, complex and ineffective antibiotic combinations have been tried with success rates below 50%. As a result, there is an urgent need to improve therapeutic options for these infections. Current treatment recommendations for MABC infection usually requires a single ?-lactam, either the cephalosporin, cefoxitin, or the carbapenem, imipenem, to be combined with other drug classes, e.g. clarithromycin and amikacin. Recent studies and our preliminary results demonstrated that combining two ?-lactams and/or a ?-lactamase inhibitor could be a successful strategy to treat MABC infections. Our studies showed dual-?-lactams (ceftazidime-imipenem or ceftazidime-ceftaroline) had the greatest synergic effects against clinical MABC in vitro and in THP-1 cells, independent of ?-lactamase inhibition with avibactam. These results provide a compelling scientific basis for our proposal to develop highly active and targeted dual-?-lactam combinations against MABC infections. As ?-lactam antibiotics primarily target peptidoglycan synthesis, we will construct novel conditional peptidoglycan remodeling enzyme repressor mutants to interrogate the molecular mechanisms underlying dual-?-lactam synergy, and to probe promising dual-?-lactam pairs against MABC infections (Aim 1). As such, we will examine the interactions between various peptidoglycan remodeling enzymes and a battery of ?-lactams, and build up a gene-compound interaction matrix of dual-?-lactam effects. Transcriptional analysis of dual-?-lactams will be used as a complimentary approach to reveal additional targets responsive to dual-?-lactam treatment (Aim 1). The efficacy of putative ?-lactam combinations will be examined against isolates collected in a well-established MABC clinical collection from over 60 cystic fibrosis clinical centers across the US (Aim 2). Spontaneous mutants conferring dual-?-lactam resistance and induced resistance mutants will be subjected to comparative genomic and RNAseq analysis to identify the resistance mechanisms. MABC peptidoglycan enzyme (e.g. ?-lactamase and transpeptidases) kinetic and hydrolysis assays will then be used to interrogate the enzymatic mechanism of ?-lactams or a ?- lactamase inhibitor against MABC infections. Lastly, we will use the state-of-art hollow fiber infection model (HFIM) and mouse models to test the preferred combinations of ?-lactams and their optimal doses, supported by pharmacokinetic (PK) and pharmacodynamic (PD) analyses (Aim 3). At the conclusion of this project, we will have developed novel dual-?-lactam combination regimens against MABC, and unraveled the molecular mechanism underlining the synergistic effects. In addition, the effectiveness the dual-?-lactams against the MABC isolates across the U.S. will be documented. The genomic output of this study will serve the basis for future work on MABC drug development, rapid diagnostics and infection control measures. The knowledge regarding peptidoglycan enzyme and ?-lactam interaction, and the repressor and induced mutant strains will be a major resource for the MABC research community.